Pre-mRNA splicing is the process in eukaryotes in which newly transcribed RNA is processed to remove intronic sequences. Splicing is highly regulated and enables the production of different mRNAs and proteins from the same gene. This is important to provide increased complexity during evolution. Alternative splicing is regulated by proteins (i.e., trans-acting proteins) which bind to regulator elements (i.e., cis-acting elements). Cis-acting elements may be located either close to or more distant from the splice sites. These include the polypyrimidine tract, branchpoints, and loosely defined regulatory elements present in either exons (exonic splicing enhancers (ESEs) and exon splicing silencers ESSs) or introns (intronic splicing enhancers (ISEs) and intronic splicing silencers (ISSs)) (reviewed in [2]). In many human genetic diseases, DNA mutations can cause aberrant splicing resulting in partial or complete disruption of protein function. Various consequences of splicing mutations can be envisioned including exon skipping, exon inclusion, intron retention, utilization of a nearby cryptic splice site, or generation of a novel splice site.
Alternative splicing often leads to more than one species of mRNA being produced from a single genetic allele. In addition to the “alternatively/aberrantly spliced” variant being produced, there is often a small amount of the wild-type mRNA produced, which is termed leaky wild-type splicing. The extent of this leaky wild-type splicing can have a predictive factor for the disease severity resulting from a splicing mutant.
Mutations and polymorphisms affecting pre-mRNA splicing are difficult to predict due to the complex mechanism of splicing regulation. Many DNA mutations are known, however the effect of these mutations on splicing is largely unknown. A number of splicing prediction programs exists [3-6], but they may produce different predictions for the same mutation or polymorphism, obscuring data interpretation. Furthermore, when weakening of a splice site is likely from in silico predictions, the effect on splicing is even more difficult to predict. Diagnostic methods often involve sequencing of the exons and a small part of the introns only. This may lead to the detection of a mutation in an intron that may affect splicing. Exonic mutations are often investigated only for their effect on protein translation. However, certain exonic mutations may also affect splicing. Sequencing of the remaining part of the introns is often not performed, also because introns can be very large in size. Intronic mutations can affect splicing, even at large distances. For example, they can create a cryptic splice site, affect RNA structure, or affect ISSs or ISEs. Promoters and UTRs are also not sequenced in diagnostics. Mutations in promoters may affect mRNA expression by changing the efficiency of RNA polymerase II-directed transcription. Mutations in UTRs may affect mRNA stability, polyadenylation, and they may interfere with regulation by micro RNAs. Exonic mutations can be studied by introducing the mutation in a cDNA and testing the effect on protein activity in a transient transfection assay, however this requires prior knowledge of the mutation. If such mutation is unknown, one cannot perform the functional assay. Effects on splicing can be determined after identification of a mutation, followed by region-specific PCR analysis. However, this requires prior identification of the mutation. This approach falls short if the mutation is not found (e.g. because it lies outside the regions normally analyzed by sequencing). In addition, it is very difficult to predict whether a mutation will affect splicing, and if so, what the outcome will be.
For example, perfect skipping of an exon while the reading frame is unchanged may generate a truncated protein with significant residual activity, while a change of the reading frame results in a premature termination codon leading to mRNA degradation via the Nonsense Mediated Decay (NMD) pathway.
Therefore, a need exists for a generic assay to systemically identify and characterize the effects of sequence variants on splicing also in the absence of mutational data. Furthermore, there is a need for an assay that may identify and characterise mutations affecting splicing and mRNA expression. In addition there is a need for method for identifying sequences that affect pre-mRNA splicing for therapeutic use.